The objective of this core TR&D project is to further improve methods for 3-dimensional immunolabeling, utilizing semithin cryosections for intermediate voltage electron microscopy (IVEM) by addressing issues such as probe penetration, signal enhancement, section quality and contrasting of unlabeled structures. Because of the penetration problems associated with colloidal gold, we have moved away from this approach and focused on the use of photo-oxidation of eosin on thick cryosections to label inaccessible antigens. We are using this approach to study 1) the distribution of ryanodine receptor isoforms in chick muscle, in collaboration with Dr. John Sutko; 2) the distribution of gap junction isoforms in liver with Dr. Gina Sosinksy and 3) the distribution of the purimidine track binding protein in the nucleus with Drs. Sui Huang and David Spector. Briefly, this technique involves cutting 3-5 urn thick cryosections, immunolabeling the antigen of interest using eosinconjugated reagents, photoconverting the section, embedding the section in epoxy and sectioning for IVEM. The labeling goes throughout the depth of the section and provides excellent resolution. At the same time as we are pursuing the photo-oxidation work, we are also looking into alternative fixation schemes to increase the penetration of reagents. We performed some preliminary work on this two years ago and recently we have utilized fixation with imidates, a non-cross linking fixative, to increase the penetration of labeling into cultured human pancreas cells. This work was performed in collaboration with Dr. Vincenzo Ciruli. Dr. Ciruli was interested in investigating the 3-dimensional organization of cell adhesion molecules in this system but was unable to obtain sufficient penetration of immunolabeling reagents to address this question. Our initial work with the non-cross linking fixative was highly encouraging and we will be directing more effort in this direction in the near future.